Rapid PCR amplification of minimal enediyne polyketide synthase cassettes leads to a predictive familial classification model

Abstract

A universal PCR method for the rapid amplification of minimal enediyne polyketide synthase (PKS) genes and the application of this methodology to clone remaining prototypical genes from producers of structurally determined enediynes in both family types are presented. A phylogenetic analysis of the new pool of bona fide enediyne PKS genes, consisting of three from 9-membered producers (neocarzinostatin, C1027, and maduropeptin) and three from 10-membered producers (calicheamicin, dynemicin, and esperamicin), reveals a clear genotypic distinction between the two structural families from which to form a predictive model. The results from this study support the postulation that the minimal enediyne PKS helps define the structural divergence of the enediyne core and provides the key tools for generating enediyne hybrid genes/molecular scaffolds; by using the model, a classification is also provided for the unknown enediyne PKS genes previously identified via genome scanning.

Fig. 1.

The naturally occurring enediynes. (a) The 9-membered enediynes include neocarzinostatin (1), C1027 (2), maduropeptin (3), kedarcidin (4), and N1999A (5). (b) The 10-membered family includes calicheamicin (6), dynemicin (7), esperamicin (8), namenamicin (9), and shishijimicin (10). The enediyne core structures are highlighted in black.

Fig. 2.

(A) Schematic representation of primer design for PCR-based amplification of enediyne PKS (PKSE) fragments. The PKSE and associated genes shown constitute the minimal PKSE cassettes from the 1, 2, and 6 biosynthetic loci, respectively, which are conserved among all enediyne gene clusters known to date. (B) The amino acid motifs used for the design of the degenerate primers A–H, the DNA sequences of which are illustrated in Table 1. The numbers between the motifs indicate the distance in amino acids. The noncoding regions between ORFs, represented by a slash, are <10 nt. (C) Amplified products using the PCR-based approach from genomic DNA isolated from the 3-, 7-, and 8-producing organisms to give madE, dynE, and espE, respectively.

Fig. 3.

Domain organization and sequence comparison between bona fide PKSs for 9-membered (1–3) and 10-membered (6–8) enediyne core structures. Domain designations: KS, ketosynthase; AT, acyltransferase; ACP, putative acyl carrier protein; KR, ketoreductase; DH, dehydratase; TD, terminal domain (possibly containing a phosphopantetheinyl transferase).

Fig. 4.

A phylogenetic comparison of bona fide enediyne minimal PKSs (a) and all putative enediyne PKSs identified to date (b). PDB ID codes: Lechevalieria aerocolonigenes AerE, AAO25864; Micromonospora echinospora CalE8, AAM94794; Streptomyces cavourensis subsp. washingtonensis CavE, AAO25858; Streptomyces citricolor CirE, AAO25874; M. chersina DynE, AAN79725; Streptomyces sp. 100/Eco52 EcoE, AAO25879; A. verrucosospora EspE, AY267372; Streptomyces ghanaensis GhaE, AAO25844; Streptomyces kaniharaensis KanE, AAO25869; Kitasatospora sp. CECT 4991 KitE, AAO25848; Streptomyces macromomyceticus MacE, AAO25894; A. madurae MadE, AY271660; Micromonospora megalomicea subsp. nigra MegE, AAO25852; Micromonospora sp. 046/Eco11 MicE, AAO25884; S. carzinostaticus NcsE, unpublished; Amycolatopsis orientalis OriE, AAO25836; Streptomyces globisporus SgcE, AY048920; Streptomyces sp. 171/Eco105 StrE, AAO25889.

Fig. 5.

A unified scheme for enediyne core biosynthesis implicating the enediyne PKS (PKSE) as responsible for a common polyketide intermediate and enediyne PKS accessory enzymes, in conjunction with the PKSE, to diverge into the 9- or 10-membered enediyne core pathways. Atoms that were incorporated intact from the acyl CoA precursors to the enediyne cores 1, 7, and 8 (–17) are shown in bold red.